New methods of treatment of cancer are needed that are able to more specifically make use of the immune system to target cancer cells and, as such, avoid the side effects typical of traditional chemotherapeutic agents. In order to better understand the immune infiltrate, it is desirable to detect NK cells in patient tissue samples, for example to detect whether NK cells are infiltrating a tumor (or more generally any site of inflammation) and/or are present at the tumor periphery or otherwise in nearby tissue. This is typically done using frozen tissue samples. This is not only useful in research but can also help in the decision about what type of treatment to use, for example, by detecting whether a tissue (e.g. a tumor environment) is characterized by inflammation that includes NK cells and/or to characterize the type of immune cells present in a tissue (e.g. tumors, inflammatory disorders). The information can be valuable in order to select a treatment that is capable of modulating the activity of the available immune cells.
In addition to frozen tissue, markers can also be detected from tissue samples that have been preserved as formaldehyde (e.g. formalin)-fixed paraffin embedded (FFPE) samples. Following deparaffination, the slides are amenable to, e.g., immunohistochemical methods to detect the expression of specific proteins. The methods have been used routinely to detect tumor antigens in tumor tissue samples. Unfortunately, it is often impossible to find monoclonal antibodies that work effectively and with specificity in FFPE sections. This is believed to be due to the impact of the formalin fixation on structure of proteins. Epitopes bound by antibodies described as being specific on recombinant protein or cells are often present on other proteins when used in FFPE, rendering the antibodies non-specific. In other cases, many epitopes on native cellular protein are destroyed by formalin fixation, causing antibodies identified using recombinant protein or cells to be ineffective for staining FFPE sections.
In the field of oncology, tumor infiltrating T cells have been the subject of extensive study. Currently, multiplexed assays in FFPE sections permit study of T cell (using CD4 and CD8 as markers) and B cells (using CD20 as marker). Tumor infiltrating NK cells, however, despite reports of their existence, have received very little attention. Levi et al (2015) Oncotarget, Advance Publications 2015, page 1-9 used anti-CD56 antibody to stain tumor infiltrating cells. Typically, markers used to stain infiltrating lymphocytes do not permit straightforward detection of cells as a single lineage, leading to combination of multiple markers in an effort to estimate the types of cells present. Additionally, different cell populations express different markers as they mature. Consequently, assessing an immune profile in a tissue sample involves considerable uncertainty and/or requires ever greater number of markers to be assessed.
While antibodies to various proteins present on NK cells exist after many decades of study, such proteins are generally either have an expression that is not highly selective to NK cells, and the expression of many activating or inhibitory NK cell receptors is shared by at least some T cells or other immune cells. At the same time many receptors have not been amenable to generation of a ligand that binds with specificity in paraffin embedded sections (e.g., no specific epitope remains available following formalin fixation). As a result, there is a need for a way to unambiguously detect NK cells in paraffin embedded samples. The present invention addresses these and other needs.